Influenza nucleoprotein vaccines

ABSTRACT

The present invention is related to a fusion protein comprising a variant of a nucleoprotein antigen from Influenza strain A, B or C, and a variant of a C4bp oligomerization domain for increasing the cellular immunogenicity of the nucleoprotein antigen from Influenza. The invention is also related to nucleic acids, vectors, fusion proteins and immunogenic compositions, for their use as a vaccine or immunotherapy for the prevention and treatment of influenza disease.

RELATED APPLICATION DATA

This application is a continuation of U.S. patent application Ser. No.14/570,155, filed Dec. 15, 2014, which is a continuation of PCTapplication PCT/EP2014/055438 designating the United States and filedMar. 18, 2014, which claims the benefit of EP application number13305320.7 and filed Mar. 18, 2013 each of which are hereby incorporatedby reference in their entireties.

BACKGROUND

A need exists for improved influenza vaccines. Current vaccinestrategies against influenza focus on generating robust antibody(humoral) responses against hemagglutinins. Because of the high degreeof antigenic drift among circulating influenza strains over the courseof a year, vaccine strains must be reformulated specifically for eachinfluenza season. Although annual (or seasonal) influenza vaccines aresuccessful to varying degrees in different age categories, moreeffective protection is clearly needed particularly for the young andthe elderly. Furthermore, there is a major, permanent risk thatreassortant viruses will evolve which have acquired very different HA(hemagglutinin) genes in a process known as “antigenic shift”. Thiswould create a public health emergency, as current influenza vaccinesrely essentially on the HA antigen.

Influenza is an enveloped, single-stranded, negative-sense RNA virus inthe Orthomyxoviridae family of viruses, divided into 3 major types: A,B, and C. Influenza A viruses infect a wide variety of animals,including humans, birds, pigs, horses, bats and many others, althoughthe tropism of any particular influenza virus is generally highlyadapted to a particular host. Influenza B viruses infect a smallernumber of species, namely humans and seals, but are still a substantialcause of annual influenza epidemics. Most human influenza infections arecaused by influenza A or B; influenza C viruses, which infect humans andpigs, rarely account for serious human infections or epidemics (Lamb).

The current inactivated influenza virus vaccines induce antibodies thatprotect against closely related virus strains. Currently licensedvaccines mainly induce strain--specific neutralizing antibodies againsthemagglutinin (HA), the main antigenic determinant on the surface of thevirus, which is highly immunogenic, and can prevent disease caused byinfection with a matching virus strain. However, HA has substantialantigenic variation which excludes its use alone in a vaccine designedto provide broad protection. For this reason, alternative vaccinestrategies that generate protective responses directed against lessvariable targets are of great interest.

Natural infection with influenza A virus induces both humoral andcellular immunity. Long-lasting cellular immunity is directedpredominantly against conserved, internal viral proteins, such as thenucleoprotein (NP). NP antigen is immunogenic in humans followingnatural infections, but the cytotoxic T lymphocytes that are inducedhave a short life-span (McMichael a, McMichael b).

Cellular immunity against NP is valuable, as it is directed againstdifferent variants of NP epitopes, and NP-targeting DNA vaccines haveinduced cross-protective immunity in animals (Schotsaert).

The nucleoprotein (NP) antigen has long been recognized as a highlyconserved antigen: even the most divergent influenza A strains share 90%identity in the NP proteins they encode (Gorman, Xu). Antigenic changesto NP are rare and only occur to a minor extent (Staneková).

PRIOR ART

Use of the Nucleoprotein as an Antigen in Vaccines

The use of influenza nucleoprotein as an antigen was described in the1980s (Wraith). Cellular immune responses in mice against NP are capableof inducing immunity, and notably of producing cross-protection againstdivergent type A viruses. It was shown that immunization of mice with NPpurified from a H3N2 virus could provide substantial protection (75%)from a lethal heterologous (H1N1) challenge, but it did not preventinfection.

DNA vaccines using the NP gene have been known for twenty years: theywere used in the first “proof of concept” experiments for DNAvaccination itself (Ulmer).

The expression of NP from a viral vector was first demonstrated in the1980s (Yewdell 1985), and immunization with this vector was associatedwith an improved generation of cytotoxic T lymphocytes against diverseinfluenza A, but not B, strains, in comparison with DNA vaccines. Since,it has been shown that immunization of mice with an MVA vectorexpressing the PR8 nucleoprotein protected them against low dosechallenges by heterosubtypic influenza viruses (Altstein). Morerecently, a viral vector encoding the NP protein fused to the M1 proteinhas been used to immunize humans (Lillie, Berthoud, Antrobus). Thesestudies showed notably that cellular immune responses to NP can besubstantially boosted in older humans (Antrobus), when humoral responsesare declining through immunosenescence.

Secretion of the Nucleoprotein

Some studies have suggested that the NP protein is primarily located inthe nucleus, reducing the immunogenicity of such DNA vaccines(Staneková).

Improved cellular immune responses against NP can be obtained by forcingthe secretion of NP, for example by fusing a tPA signal peptide to theNP gene (Luo), by formulation of the DNA (Greenland, Sullivan) and bythe use of electroporation (Laddy) to improve DNA delivery.

Monomeric Influenza Nucleoproteins

The preferred use of monomeric antigens in fusion with C4bpoligomerisation domains was described in the patent application WO2005/014654. But the risk in using monomerised antigens is theirdecreased immunogenicity. This was demonstrated by Bachmann andcolleagues with the glycoprotein G of Vesicular Stomatitis Virus(Bachmann 1993), and for the influenza antigen Neuraminidase, or NA, byFiers and colleagues (Fiers 2001). It is to be expected that decreasingor removing higher order structure from influenza nucleoproteins woulddecrease their immunogenicity.

A number of mutations have been shown to transform the influenzanucleoprotein, which naturally oligomerizes, into a monomeric form (Ye2006). Monomeric versions of NP described in this 2006 paper wereconfirmed to be monomeric in more recent papers (Tarus, Ye 2012). Thetwo point mutations described in these papers which render monomeric thenucleoprotein of influenza A, are conserved in the nucleoproteins ofinfluenza B and C strains (see FIG. 3 in Nakada). Therefore the samepoint mutations could be introduced in nucleoproteins of influenza B andC strains, in order to render monomeric these other influenzanucleoproteins. But no studies of the immunogenicity of the monomericnucleoproteins were carried out.

The major technical problem in preparing influenza vaccines with the NPantigen is inducing strong and durable cellular immune responses. The‘cellular immune response’ is an immune response that does not involveantibodies but rather involves the activation of antigen-specificT-lymphocytes, and especially cytotoxic T lymphocytes, and the releaseof various cytokines in response to an antigen. CD4 cells or helper Tcells provide protection against different pathogens by secretingcytokines that activate the immune response. Cytotoxic T cells (CD8)cause death by apoptosis of pathogens without using cytokines.

Although debate remains as to whether CD4 or CD8 responses against NPare more important for protection (Epstein), there is a consensus thatcellular, rather than humoral, responses to the nucleoprotein are thekey to the protection that this antigen can induce (Thomas). Vaccinesthat provide protection by eliciting a strong cytotoxic T cell responsemay be useful when T cell epitopes are derived from the highly conservedNP protein (Epstein; Roy). Cellular immune responses, mediated by Tlymphocytes, mainly function by recognizing influenza virus-infectedcells, by inhibiting viral replication and by accelerating virusclearance.

The specific T cells involved in conferring immunity include both CD4+and CD8+ T cells, and often exert their functions through the action ofsecreted cytokines and cytolytic activity, respectively. InfluenzaNP-specific CD8+ CTL in particular could play important roles inheterosubtypic protective immunity against a lethal influenza viruschallenge in mice (Gschoesser), including clearance of the influenzavirus from the upper respiratory mucosal surfaces (Mbawuike), promotingsurvival and recovery after challenge (Epstein). An optimal NP-basedvaccine would improve both CD4 and CD8 cellular responses.

This patent application provides methods for improving cellular immuneresponses to influenza virus nucleoproteins.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is related to a method for increasing theimmunogenicity, and particularly the cellular immunogenicity, of the NPantigens from Influenza viruses, by fusing at least one variant of theNP antigen from Influenza strains A, B or C to a variant of the chickenC4bp oligomerization domain as carrier protein.

The present invention is in particular related to a fusion protein,comprising at least one monomeric variant of the NP antigen fromInfluenza strain A, B or C, and a carrier protein IMX313 having thesequence as shown in SEQ ID NO: 1, such as described in the patentapplication WO2007/062819.

The present invention is in particular related to a fusion protein,comprising the monomeric variant of the NP antigen from Influenza strainA presenting the E339A and R416A point mutations as shown in SEQ ID NO:2, and a variant of IMX313 carrier protein having a C-terminalsubstitution of at least one positively charged peptide having thesequence ZXBBBBZ wherein (i) Z is any amino acid or is absent, (ii) X isany amino acid and (iii) B is an arginine (R) or a lysine (K), as shownin SEQ ID NO: 3, such as described in the patent applicationPCT/EP2013/076289 filed on Dec. 11, 2013. A preferred variant of IMX313carrier protein does not induce antibodies which cross-react withprotamine.

The present invention is in particular related to a fusion protein,comprising a monomeric variant of the NP antigen, and a modified carrierprotein IMX313T or IMX313P, as shown respectively in SEQ ID NO: 4 andSEQ ID NO: 5.

The present invention is also related to an immunogenic compositioncomprising a DNA sequence in a plasmid or a viral vector, furthercomprising a signal peptide, such as tPA, as shown in SEQ ID NO: 6.

The present invention is also related to a recombinant DNA sequencecoding for said fusion proteins.

The present invention is also related to an immunogenic compositioncomprising a DNA sequence encoded by a plasmid or a viral vector, or afusion protein, further comprising vaccine adjuvants or nucleic acidligands for intracellular TLRs, as described in the patent applicationPCT/EP2013/076289 filed on Dec. 11, 2013.

The present invention is also related to a DNA plasmid, a viral vector,a fusion or an immunogenic composition, for its use as a vaccine or animmunotherapy as a method of prevention or treatment of the influenza.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified methods and may, of course, vary. In particular, the presentinvention is related to fusion proteins comprising at least onenucleoprotein antigen from Influenza, and is not limited to a specificinfluenza nucleoprotein.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.However, publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols, reagents and vectors that arereported in the publications and that might be used in connection withthe invention.

Furthermore, the practice of the present invention employs, unlessotherwise indicated, conventional protein purification and molecularbiological techniques within the skill of the art. Such techniques arewell known to the skilled worker, and are explained fully in theliterature. In the claims that follow and in the consecutive descriptionof the invention, except where the context requires otherwise due toexpress language or necessary implication, the word “comprise”,“contain”, “involve” or “include” or variations such as “comprises”,“comprising”, “containing”, “involved”, “includes”, “including” are usedin an inclusive sense, i.e. to specify the presence of the statedfeatures but not to preclude the presence or addition of furtherfeatures in various embodiments of the invention.

The following terms are defined for a better understanding of theinvention:

Influenza viruses are of three types, A, B and C. This classificationwas initially serological: antisera to the influenza virus Anucleoprotein cross-react with the nucleoprotein of other A classviruses, but not with those of B class or C class viruses. Influenza Aviruses are further classified into subtypes, based on the serologicalcross-reaction of their hemagglutinin (H) and neuraminidase (N)glycoproteins.

By “Influenza nucleoproteins” are meant the nucleoproteins of all threetypes (A, B and C) of the Influenza viruses.

A “carrier protein” designates generally a protein to which antigens areconjugated or fused and thereby rendered more immunogenic. Here the termis used specifically in the meaning of a protein carrying an antigen.The function of the protein is to increase the immunogenicity of saidantigen to which it is conjugated or fused.

A “variant of NP” designates all the proteins having a sequence with atleast 90% of identity of the wild type version of influenzanucleoproteins from Influenza strains A, B and C.

A “variant of the chicken C4bp oligomerization domain” is a variant ofthe C4bp domain of the SEQ ID NO: 1 described in the patent applicationsWO2007/062819, and PCT/EP2013/076289 filed on Dec. 11, 2013, bothreferences being incorporated herein by reference, particularly afragment of at least 48 contiguous amino acids and/or having at least70% amino acid sequence identity of said SEQ ID NO: 1 described in thepatent applications WO2007/062819.

A “variant of IMX313 carrier protein” is described in the patentapplication PCT/EP2013/076289 filed on Dec. 11, 2013.

Protamine consists of a group of heterogenous peptides with an averagemolecular weight of 4500 Daltons, obtained from fish. Approximately 67%of the amino acid composition of protamine is arginine. It has long beenused to formulate insulin (in Neutral Protamine Hagedorn), or toneutralize heparin.

The term “fusion protein” designates a recombinant protein,non-naturally existing, comprising two domains from different originsthat have been fused. More precisely, in the invention, the fusionprotein comprises an influenza nucleoprotein antigen fused to a carrierdomain variant of the chicken C4bp oligomerization domain, particularly‘IMX313T’ or ‘IMX313P’. Fusion has the advantage of creating ahomogenous product. More formally, the “conjugation” can be described asgenetic: the DNA encoding the pro-immunogenic carrier protein is splicedto the DNA encoding the antigen. The antigen can be fused to the N- orC-terminal of the carrier protein.

The invention is related to an immunogenic composition, comprising atleast one variant of an Influenza nucleoprotein antigen and a variant ofa C4bp oligomerization domain, and eliciting a increased cellular immuneresponse against the Influenza nucleoprotein antigen.

According to the present invention, the nucleoprotein fused to thecarrier protein variant of the chicken C4bp oligomerization domain,particularly IMX313T or IMX313P, can be a nucleoprotein from any type(A, B or C) of the Influenza viruses.

The nucleoprotein antigen can be fused to the N- or C-terminal of thecarrier protein, particularly IMX313T or IMX313P.

According to the invention, at least one nucleoprotein is fused to onecarrier protein, particularly IMX313T or IMX313P; however, two or morenucleoproteins, identical or different, can be fused to the same carrierprotein.

According to a preferred aspect of the invention, the nucleoproteinantigen fused to the variant of the chicken C4bp oligomerization domain,particularly IMX313T or IMX313P, is a monomeric antigen. Indeed, it isadvantageous to use monomeric antigens, as described in the patentapplication WO 2005/014654, provided that monomerization does notdiminish their immunogenicity. Moreover, NP forms a trimer in crystals(Ye 2006) and other oligomers in vivo (Arranz, Moeller). Fusion of atrimeric or oligomeric protein to a heptameric protein such as IMX313Tor IMX313P risks the creation of steric clashes. On the other hand,monomeric forms of naturally oligomeric proteins have diminishedimmunogenicity (Fiers).

To obtain monomeric nucleoprotein antigen, the man skilled in the artknows different point mutations that can be introduced into the proteinsequence of the NP antigen, to induce its monomerisation. In particular,the NP antigen presents at least one of the two following pointmutations: E339A and R416A.

In an embodiment of the invention, the NP antigen is from the Influenzastrain A.

In a preferred embodiment of the invention, the NP antigen comprisesboth point mutations E339A and R416A, and is therefore monomeric.

In another embodiment of the invention, the NP antigen presents thepolypeptidic sequence as shown in SEQ ID NO: 2.

Increased cellular immune responses to antigens expressed from DNAvaccines have previously been obtained by fusing the gene of the antigento a gene encoding the IMX313 (Spencer). Advantageously, variants ofthis domain presenting a C-terminal substitution of at least onepositively charged peptide having the sequence ZXBBBBZ wherein (i) Z isany amino acid or is absent, (ii) X is any amino acid and (iii) B is anarginine (R) or a lysine (K), as shown in SEQ ID NO: 3, which enables animproved immune response to an antigen when fused to said antigen, suchas described in the patent application PCT/EP2013/076289 filed on Dec.11, 2013. A preferred variant of IMX313 carrier protein does not induceantibodies which cross-react with protamine.

Particular improved variants, called IMX313T and IMX313P, have beenrecently described in the patent application PCT/EP2013/076289 filed onDec. 11, 2013. Their peptide sequences are the following:

SEQ ID NO: 4 KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRK- LFLEIQKLKVELQSPRRRRSSEQ ID NO: 5 KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRK- LFLEIQKLKVEGRRRRRS

In another embodiment of the invention, the fusion protein comprises aNP antigen which comprises a signal peptide. Several studies havesuggested that the NP protein is primarily located in the nucleus, whichcould potentially reduce the immunogenicity of such DNA vaccines.Therefore, enabling the secretion of NP antigen, by adding a signalpeptide, is desirable. In particular, said signal peptide is the tPA(tissue plasminogen activator) secretory signal peptide as described in(Luo).

In a specific embodiment of the invention, the NP antigen is monomericand comprises a signal peptide.

In another embodiment of the invention, the NP antigen comprises bothmutations E339A and R416A, and the signal peptide tPA. As shown in theexamples, the fusion protein comprising IMX313T and the monomeric NPantigen comprising a signal peptide induces, when injected as a DNAvaccine in mice, a stronger Th1 response (IgG2a) than Th2 response (FIG.13). The consensus among immunologists is that Th1 responses arepreferable to Th2 responses. However methods for improvingpreferentially Th1 responses to an antigen, without the use of adjuvantsdeveloped for this purpose, are not known in the art. In the examplesbelow, it is shown that the fusion of IMX313T or IMX313P to influenzanucleoprotein antigens preferentially improves Th1 responses.

The invention is also related to a fusion protein comprising any carrierprotein comprising a coiled-coil domain, and at least one nucleoprotein(NP) antigen from Influenza. In particular, said nucleoprotein antigenis monomeric.

The present invention is also related to a nucleic acid encoding for afusion protein such as described above, and in particular:

-   -   coding for a fusion protein comprising a NP antigen and IMX313T        or IMX313P;    -   coding for a fusion protein comprising a monomeric NP antigen        and IMX313T or IMX313P;    -   coding for a fusion protein comprising a NP antigen comprising a        signal peptide and IMX313T or IMX313P;    -   coding for a fusion protein comprising a monomeric NP antigen        comprising a signal peptide and IMX313T or IMX313P.

As preferred embodiments, said nucleic acid codes for a fusion proteincomprising a monomeric NP antigen from Influenza A. As preferredembodiments, said nucleic acid codes for a fusion protein which does notinduce antibodies cross-reacting with protamine. In particular, saidnucleic acids present the sequences as shown in SEQ ID NO: 6 and SEQ IDNO: 7.

The present invention is also related to a vector comprising the nucleicacid presented above, and genetic elements such as promoters andenhancers to ensure the expression of the DNA cassette in host cells.

The present invention is also related to an immunogenic compositioncomprising:

-   -   a fusion protein or a nucleic acid or a vector as presented        above, and    -   nucleic acid ligands for intracellular TLRs, and/or any other        vaccine adjuvants.        Toll-Like Receptors (TLRs)

Cells of the innate immune system detect pathogens through a limited setof germ-line encoded receptors. These innate immune receptors recognizea series of conserved molecular structures expressed by pathogens, thePAMPs (pathogen associated molecular patterns).

These pathogen-derived molecules generally represent complex moleculesthat are very specific for a set of pathogens. TLRs represent a set ofimmune pattern recognition receptors able to alert the immune systemimmediately after infection by a pathogen. They play an important roleas pivotal components between innate and adaptive immunity and are ableto scent out many pathogens ranging from viruses to parasites. The firstcharacterized TLR, called Toll, was shown to be responsible foranti-fungal responses in the adult Drosophila fly and 10 humanequivalents involved in pathogen recognition have been identified todate. TLRs can be classified into different groups based on theirlocalization and the type of PAMPs they recognize. TLRs 1, 2, 4, 5 and 6are principally expressed on the cell surface, where they recognizemostly bacterial products, while TLRs 3, 7, 8 and 9 are localized inintracellular compartments and recognize mostly viral products andnucleic acids.

Intracellular Toll-Like Receptors

Besides, to improve methods of immunization, it is also of greatimportance to limit signaling through TLR receptors. Toll-like receptors(TLRs) are a class of protein that play a key role in the innate immunesystem. Once microbes have breached physical barriers of organisms, theyare recognized by TLRs. The recognized features from microbes includedouble-stranded RNA of viruses, unmethylated CpG site islands ofbacterial and viral, and certain RNA and DNA molecules.

There is substantial interest in such nucleic acids as they are ligandsfor a class of Toll-Like Receptors (hereafter TLRs), and notably forTLR3, TLR7, TLR8, TLR9 and TLR13 (Blasius and references therein). Theseare sometimes classed as the “Intracellular Toll-like Receptors”, but atleast TLR3 is also present on some cell surfaces. TLR3 is expressed on avariety of epithelial cells including airway, uterine, corneal, vaginal,cervical, biliary and intestinal epithelial cells, and these cellsappear to express TLR3 on their cell surfaces (Akira)

The importance of limiting signaling through these receptors, andnotably the TLR3 receptor, is dose-dependent. Binding nucleic acidligands tightly to the antigen is thus essential, to prevent theirbinding to TLRs in the absence of the antigen. Tightly boundintracellular TLR ligands are therefore highly preferred overformulations in which binding is less tight. Therefore, the man skilledin the art is looking for antigenic compositions able to bindefficiently TLR ligands, so that they are not separated from the antigenbefore the antigen arrives in the cells where it will trigger an immuneresponse, with the goal of diminishing the potential adverse effectsmediated by the binding of the ligands to TLR receptors elsewhere.

In the present application, and in particular in examples, the followingintracellular TLR ligands have been used:

-   -   For TLR3: poly I:C being a duplex of a polynucleotide of        polyinosinic acid hybridized to polycytidylic acid, an analogue        of double-stranded RNA. The chain length was twenty nucleotides        for each strand.    -   For TLR7: an oligonucleotide, called ssRNA40, with the sequence        5′ GsCsCsCsGsUsCsUsGsUsUsGsUsGsUsGsAsCsUsC 3′ where “s”        represents a phosphothioate linkage (SEQ ID NO: 8);    -   For TLR9: an oligonucleotide called ODN1826 with the sequence:        5′ tccatgacgttcctgacgtt 3′ (SEQ ID NO: 9).

In a specific aspect of the invention, the immunogenic compositioncomprises:

-   -   a fusion protein or a nucleic acid or a vector as presented        above, and    -   poly I:C.

The invention is also related to a fusion protein such as describedabove, for its use as a vaccine for the prevention and treatment ofinfluenza disease. Said influenza vaccine can be used for multipleapplications:

-   -   prevention of seasonal influenza;    -   prevention in a pandemic situation;    -   ‘universal’ prevention, i.e. a vaccine immunizing against all        types of influenza viruses;    -   immunotherapy of all types of influenza.

Methods of prevention or treatment of the influenza can be performed,with specific vaccines according to the invention, in human or animalbodies. The man skilled in the art knows how to adapt the compositionsof vaccines for each specific application and specific patients.

The invention is also related to a nucleic acid such as described above,for its use as a DNA vaccine for the prevention of influenza disease.

The invention is also related to a vector such as described above, forits use as a viral vaccine for the prevention of influenza disease.

The invention is also related to an immunogenic composition such asdescribed above for its use as a vaccine for the prevention of influenzadisease.

The invention is also related to a method for increasing the cellularimmune response to the nucleoprotein antigen of influenza, comprisingthe fusion of this antigen to a carrier protein IMX313T or IMX313Phaving the sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 5.

In another embodiment of the invention, the fusion protein or thenucleic acid or the vector or the immunogenic composition such asdescribed previously is used in immunotherapy of influenza disease.

DRAWINGS

FIG. 1: map of the parental plasmid pcDNA3 NP—This plasmid and itsderivatives, constructed as described in the Examples, were used for DNAvaccination.

FIG. 2: Comparison of total T cells secreting IFN-γ in response toimmunization with plasmids encoding NP, or NP fused to IMX313.

FIG. 3: Comparison of CD8 and CD4 T cells secreting IFN-γ in response toimmunization with a plasmid encoding NP or a plasmid encoding NP fusedto IMX313.

FIG. 4: Comparison of IgG antibody responses to recombinant NP inducedby DNA plasmids encoding either NP or NP fused to IMX313

FIG. 5: Comparison of IgG antibody subclass responses to recombinant NPinduced by DNA plasmids encoding either NP or NP fused to IMX313.

FIG. 6: Comparison of total T cell responses to plasmids encoding NP,monomeric NP (NPm), monomeric NP fused to IMX313 (NPm-IMX313) andmonomeric NP fused to IMX313T (NPm-IMX313T).

FIG. 7: Comparison of CD8+ and CD4+ T cell responses to plasmidsencoding NP, monomeric NP (NPm), monomeric NP fused to IMX313(NPm-IMX313) and monomeric NP fused to IMX313T (NPm-IMX313T).

FIG. 8: Comparison of IgG antibody responses, measured by ELISA usingrecombinant NP, to plasmids encoding NP, monomeric NP (NPm), monomericNP fused to IMX313 (NPm-IMX313) and monomeric NP fused to IMX313T(NPm-IMX313T).

FIG. 9: Comparison of IgG antibody subclass responses, measured usingrecombinant NP, to plasmids encoding NP, monomeric NP (NPm), monomericNP fused to IMX313 (NPm-IMX313) and monomeric NP fused to IMX313T(NPm-IMX313T).

FIG. 10: Influence of the secretion, by the tPA signal peptide, on thevarious NP fusion proteins. Total T cells were measured by IFNγ ELISpotscomparing NP, secreted NP (tPA-NP), secreted monomeric NP (tPA-NPm),secreted NP fused to IMX313 (tPA-NP-IMX313), secreted monomeric NP fusedto IMX313 (tPA-NPm-IMX313), and secreted monomeric NP fused to IMX313T(tPA-NPm-IMX313T).

FIG. 11: Influence of the secretion, by the tPA signal peptide, on theCD8+ and CD4+ responses to various NP fusion proteins, measured by IFNγELISpots comparing: NP, secreted NP (tPA-NP), secreted monomeric NP(tPA-NPm), secreted NP fused to IMX313 (tPA-NP-IMX313), secretedmonomeric NP fused to IMX313 (tPA-NPm-IMX313), and secreted monomeric NPfused to IMX313T (tPA-NPm-IMX313T).

FIG. 12: Influence of the secretion, by the tPA signal peptide, on theIgG responses to various NP fusion proteins, measured by ELISAscomparing: NP, secreted NP (tPA-NP), secreted monomeric NP (tPA-NPm),secreted NP fused to IMX313 (tPA-NP-IMX313), secreted monomeric NP fusedto IMX313 (tPA-NPm-IMX313), and secreted monomeric NP fused to IMX313T(tPA-NPm-IMX313T).

FIG. 13: Influence of the secretion, by the tPA signal peptide, on theIgG subclass responses to various NP fusion proteins, measured by ELISAscomparing: NP, secreted NP (tPA-NP), secreted monomeric NP (tPA-NPm),secreted NP fused to IMX313 (tPA-NP-IMX313), secreted monomeric NP fusedto IMX313 (tPA-NPm-IMX313), and secreted monomeric NP fused to IMX313T(tPA-NPm-IMX313T).

FIG. 14: Fusion of nucleoprotein to IMX313T increases the immunogenicityof NP to the same extent as the formulation of NP in the oil-in-wateradjuvant AddaVax (Invivogen); and the use of AddaVax with theNPm-IMX313T fusion protein shows a synergistic effect.

FIG. 15: Analysis of the results shown in FIG. 14 after separation ofCD4 and CD8 cells. The synergistic effect of AddaVax with theNPm-IMX313T protein is seen both in CD4 responses and the CD8 responses.

FIG. 16: IgG responses to nucleoprotein. Fusion of nucleoprotein toIMX313T, in the absence of the adjuvant AddaVax, showed no significantchange in IgG titres compared to NP. But in the presence of AddaVax, thefusion protein is significantly more immunogenic than the nucleoprotein.

FIG. 17: Comparison of IgG antibody subclass responses, measured usingrecombinant NP, following immunization with NP or NPm-IMX313T, with orwithout AddaVax. As seen in Table 4, NP, with or without Addavax,induced a Th1 response. But the fusion protein NPm-IMX313T, with orwithout AddaVax, further polarized the IgG response towards Th1.

FIG. 18: SDS-PAGE analysis of the recombinant proteins used forimmunisations. Lane 1: molecular weight markers (New England Biolabs);lane 2: recombinant NP (Imgenex); lane 3: purified NP; lane 4: purifiedNPm-IMX313T.

FIG. 19: SDS-PAGE analysis of the recombinant NPm-IMX313P proteins. Lane1: purified NP; lane 2: purified NPm-IMX313T; lane 3: purifiedNPm-IMX313P; lane 4: molecular weight markers (New England Biolabs).

FIG. 20: IgG responses to Protamine or to IMX313P, after immunization ofmice with IMX313P protein. This shows that, although the mice produceIgG antibodies to IMX313P (and some cross-react with IMX313), noantibodies which cross-react with protamine were found.

EXAMPLES

For DNA vaccinations, the parent plasmid pcDNA3-NP, as shown in FIG. 1,was modified as described in the Examples below. The plasmids pIMX494and pIMX497 are described in the patent application PCT/EP2013/076289filed on Dec. 11, 2013.

Example 1—Insertion of IMX313 into NP Encoding Plasmids

The IMX313 coding sequence was amplified from the plasmid pIMX494 usingthe oligonucleotide primers IMX1289 (5′caatgcagaggagtacgacaatggatccaagaagcaaggtgatgctgatg 3′—SEQ ID NO: 10) andIMX1290 (5′ GTAGAAACAAGGGTATTTTTCTTtattactccttgctcagtccttgc 3′—SEQ IDNO: 11) and inserted into the plasmid pcDNA3-NP as described by Geiser.

Example 2—Insertion of the tPA Signal Peptide

The tPA signal peptide was amplified from the vector pSG2-85A (Spencer)using the oligonucleotides IMX1305 (5′cactgagtgacatcaaaatcatgGATGCAATGAAGAGAGGGC 3′—SEQ ID NO: 12) and IMX1306(5′ cgtaagaccgtttggtgccttggctagctcttctgaatcgggcatggatttcc 3′—SEQ ID NO:13) and inserted in-frame with the N-terminus of the NP coding sequencein a number of plasmids as described by Geiser.

Example 3—Creation of Two Point Mutations of NP to Render it Monomeric

The oligonucleotide primers IMX1287 (5′ ccattctgccgcatttgCagatctaagag3′—SEQ ID NO: 14) and IMX1288 (5′ CAAAAGGGAGATTTGCCTGTACTGAGAAC 3′—SEQID NO: 15) were used to amplify an internal fragment of the NP gene, andthe resulting PCR product was inserted into NP-encoding plasmids asdescribed by Geiser. Because both oligonucleotides were imperfectlymatched to the NP gene, the insertion of the PCR product generated twopoint mutations. The IMX1287 primer created the mutation E339A (GAA toGCA), whereas the IMX1288 primer created the mutation R416A in the NPgene (AGA to GCA).

Example 4—Insertion of IMX313T

The IMX313T coding sequence was amplified from the plasmid pIMX497 usingthe oligonucleotide primers IMX1289 (SEQ ID NO: 10) and IMX051 (5′GTAGAAACAAGGGTATTTTTCTTtattaggagcgacggcgacgc 3′—SEQ ID NO: 16) andinserted into the various pcDNA3-NP-derived plasmids as described byGeiser.

Example 5—DNA Immunizations with the Nucleic Acids According to theInvention

5.1. Protocol

Groups of five female BALB/C mice were immunized intramuscularly twice,14 days apart, with various plasmid DNAs, using 20 μg of each plasmidper injection. Immune responses were measured on day 28, to determinethe influence of various modifications: +/−IMX313 or IMX313T; +/−the tPAsignal peptide; +/−the monomerizing mutations.

Antigen-specific T-cell responses were measured by ELISPOTs, usingsplenocytes, on day 28. Purified spleen CD4+, CD8+ and Total T cellsisolated from the immunized mice were co-cultured with NP A Influenzapeptide (amino acids 366-374) purchased from Eurogentec. ELISPOT Assays:Flat-bottomed, 96-well nitrocellulose plates (Millititer; Millipore)were coated with IFN-γ mAb (15 μg/ml; MABTECH, Stockholm) and incubatedovernight at 4° C. After washing with PBS, plates were blocked with 10%fetal bovine serum for one hour at 37° C. 2×10⁶ cells per well werestimulated with relevant peptides at a final concentration of 2 μg/ml(NP A Influenza peptide) on IPVH-membranes coated with 15 μg/mlanti-human IFN-γ and incubated for 20 hours. After incubation, theplates were washed thoroughly with PBS to remove cells, and IFN-γ mAb(15 μg/ml of biotin, MABTECH) was added to each well. After incubationfor 2 h at 37° C., plates were washed and developed withstreptavidin-HRP (1 μg/ml; MABTECH) for one hour at room temperature.After washing, the substrate (3-amino-9-ethycarbazol (Sigma)) was addedand incubated for 15 minutes. After further washing, the red spots werecounted under the microscope.

To study the humoral immune responses, we evaluated the antibody levelsby ELISAs specific for total IgG, and separately for IgG1 and IgG2a toevaluate the relative proportions of Th1 and Th2. BALB/c mice typicallyrespond to influenza vaccines with a Th2-type immune response, which isassociated with the stimulation of IgG1 antibodies. However, the majorantibody isotype present in the sera of mice that survive viralinfections is IgG2a, which is stimulated during Th1-type immuneresponses (Huber). Stimulation of IgG2a antibodies has been associatedwith increased efficacy of influenza vaccination.

For the ELISAs, antigens were diluted to a concentration of 5 mg/ml in0.1 M sodium carbonate/bicarbonate (pH 9.6) and were then used to coatthe wells of MaxiSorb plates (Nunc-Immulon, Denmark). Twofold serialdilutions of the test sera were added to the wells, and followingwashing, bound antibodies were detected with anti-mouse IgG, oranti-mouse IgG1 or anti-mouse IgG2a (Sigma) conjugated to horseradishperoxidase. Absorbance at 490 nm was determined after o-phenylenediamine(Sigma) and H₂O₂ were added; the reactions were stopped with 1 Msulphuric acid.

Results are shown in FIGS. 2 to 5.

5.2. In preliminary experiments, we tested total T cell responses to NPinduced by DNA vaccines encoding either NP or NP fused to IMX313. TotalT cells isolated from the NP-IMX313 immunized mice showed significantlyhigher IFN γ responses compared with those of the NP immunized mice andconfirmed the ability of IMX313 to increase T cell responses.

5.3. To determine whether the IFN-γ detected in the ELISPOTs wasproduced by CD4 or CD8 T cells, we purified spleen CD4+ and CD8+ T cellsfrom the immunized mice, and these were co-cultured with an Influenza ANP peptide. A significant increase in IFN-γ production from CD8+ T cellswas detected in the group immunized with NP-IMX313. The percentage ofantigen-specific CD8+ cells producing IFN-γ was higher than thecorresponding population of CD4+ T (FIG. 3).

FIG. 3 shows that fusing the NP antigen gene to the IMX313 gene improvesboth CD4+ and CD8+ responses to the NP antigen.

5.4. We then examined the antibody response to NP after immunization and14 days after the last immunization, NP-specific IgG Ab responses weremeasured in sera. NP control mice and mice given NP-IMX313 showedmoderate NP-specific IgG Abs (FIG. 4), which were higher in the groupimmunized with NP-IMX313.

FIG. 4 shows that fusing the NP gene to the IMX313 gene improves IgGantibody responses to the NP antigen.

5.5. Sera were also examined for the presence of NP-specific IgG1 andIgG2a antibodies (representative of Th2 and Th1 types of response inBalb/C mice, respectively). NP-specific IgG1 and IgG2a antibody isotypeswere detected in the sera of the NP-IMX313 immunized mice; however serumsamples from mice given NP alone showed only low levels of IgG1 andIgG2a Ab (FIG. 5).

FIG. 5 shows the subclass distributions of the antibodies inducedagainst the NP antigen. Fusion to the IMX313 gene improved the IgG2Aresponse more than the IgG1 response, converting a Th2-biased responseagainst NP to a Th1-biased response against NP-IMX313. The results aretabulated here:

TABLE 1 Immunogenic IgG2a IgG1 Component Subclass Subclass IgG2a/IgG1 Thpattern NP 0.215 0.265 0.8 Th2 NP-IMX313 0.528 0.35 1.51 Th1

FIG. 6 shows that monomerisation of NP (NPm) improves its immunogenicityslightly (although the improvement is not statistically significant:NS); that NPm immunogenicity is further improved by fusion to the IMX313gene; and finally that fusing the monomeric NP to the IMX313T genefurther enhances NP immunogenicity. Surprisingly, monomerisation of NPdoes not decrease its immunogenicity.

FIG. 7 shows that, on analysis of the CD4+ and CD8+ responses, the samerank ordering as in FIG. 6 is seen: monomerisation of NP improves NP'simmunogenicity slightly but not significantly (NS); NP's immunogenicityis further improved by fusion to the IMX313 gene, but that the largestimprovement in NP immunogenicity is obtained by fusing the monomeric NPto the IMX313T gene.

FIG. 8 shows that the same rank ordering is seen for B cell responses aswas seen for T cell responses (both CD4+ and CD8+) in FIGS. 6 and 7.Total IgG responses against NP were higher with IMX313T than withIMX313.

FIG. 9 shows the subclass distributions of the antibodies induced by themonomeric NP antigen. As with NP, fusion to the IMX313 gene augmentedthe IgG2A responses more than the IgG1 responses, converting aTh2-biased response against NP (0.8) to a Th1-biased response againstNP-IMX313 (1.51). This reversal of a Th2 to a Th1 bias maintained byfusion to IMX313T rather than to IMX313 (1.5). Expression of IgG2aantibodies in the influenza vaccines is correlated with clearance ofvirus and increased protection against lethal influenza challenge.Increased induction of both antibody isotypes as measured by ELISA was abetter correlate for vaccine efficacy than neutralization alone (Huber).

TABLE 2 Immunogenic IgG2a IgG1 Component Subclass Subclass IgG2a/IgG1 Thpattern NP 0.215 0.265 0.8 Th2 NPm 0.4 0.363 1.1 Th1 NPm-IMX313 0.5280.35 1.51 Th1 NPm-IMX313T 0.95 0.632 1.5 Th2

Example 6—Secretion of the NP Antigen Improved its Immunogenicity

A series of NP DNA vaccine constructs containing the tissue plasminogenactivator (tPA) secretory signal sequence was made: tPA-NP, tPA-NPm,tPA-NPm-IMX313, and tPA-NPm-IMX313T. The effects of the fusion of tPA toNP on the humoral and cellular immune responses from the immunizedanimals were analyzed.

Mice immunized with tPA containing constructs showed significantlyhigher IFNγ responses compared with those of the NP immunized mice andconfirmed the ability of IMX313T and the monomerizing mutations toincrease T cell responses.

FIG. 10 shows that forcing the secretion of the NP antigen improved itsimmunogenicity (NP versus tPA-NP), whether it was monomeric or not(tPA-NP versus tPA-NPm). However, fusion to IMX313 showed that use of amonomeric version of NP was more immunogenic than use of the unmodifiedantigen (tPA-NP-IMX313 versus tPA-NPm-IMX313). And substitution ofIMX313 by IMX313T further improved the immunogenicity of NP(tPA-NPm-IMX313 versus tPA-NPm-IMX313T).

FIG. 11 shows the CD8+ and CD4+ responses to the different secretedversions of NP. The same rank ordering as in FIG. 10 is seen, and theutility of monomerising the antigen is once again pronounced when IMX313is added. As in the preceding Figures, the largest immune responses areseen when IMX313T is used rather than IMX313.

FIG. 12 shows the total IgG responses to the antigen NP and invites thesame conclusions as FIG. 11 for T cell responses: the largest responsesare seen when IMX313T is used, but secretion (NP versus tPA-NP) andmonomerisation (tPA-NP-IMX313 versus tPA-NPm-IMX313) are also importantcontributions.

Mice immunized with NP alone (as NP, tPA-NP or tPA-NPm) had no or verylow levels of anti-NP IgG antibody in their sera (FIG. 12) Miceimmunized with tPA-NP-IMX313, tPA-NPm-IMX313 or tPA-NPm-IMX313T on theother hand, showed high levels of systemic NP-specific IgG antibodyresponses; once again, the tPA-NPm-IMX313T immunized mice hadsignificantly higher (p<0.001) IgG antibody responses compared to allthe other groups of immunized mice. This shows that the combination ofall the modifications (monomerizing mutations, tPA and IMX313T) confersa significantly improved immunogenicity to the antigen compared to theparental sequence or other combinations.

FIG. 13 shows the subclass analysis of the B cell responses to NP, andillustrates that the initial Th2 bias with NP alone is reversed byIMX313 and by IMX313T. While secretion has little effect on its own (NPversus tPA-NP), monomerisation (tPA-NP-IMX313 versus tPA-NPm-IMX313) andthen the replacement of IMX313 by IMX313T (tPA-NPm-IMX313 versustPA-NPm-IMX313T) all contribute to the improved Th1 (IgG2a) versus Th2(IgG1) responses.

It is very important that tPA-NPm-IMX313T on its own improves almostequivalently Th1 and Th2 responses. Fusion of NP to IMX313 shows thatboth Th1 and Th2 responses are both increased, and there is nosignificant shift in the type of response. But with IMX313T and themonomerizing mutations combined, the Th1 response (IgG2a) starts topredominate. The consensus among immunologists is that Th1 responses arepreferable to Th2 responses (FIG. 13).

These results are tabulated here:

TABLE 3 Immunogenic IgG2a IgG1 Component Subclass Subclass IgG2a/IgG1 Thpattern NP 0.215 0.265 0.8 Th2 tPA-NP 0.27 0.31 0.85 Th2 tPA-NPm 0.3280.363 0.9 Th2 tPA-NPm-IMX313 0.528 0.35 1.51 Th1 tPA-NPm-IMX313T 0.950.632 1.5 Th1

Example 7—Production of Recombinant NPm-IMX313T Protein

A pET22-derived plasmid expressing the wild-type H1N1 NP protein ofstrain A/WSN/33 (Tarus 2012b) with a C-terminal 6-His-tag was expressedin the bacterial strain C43R. This strain was made by transformingC43(DE3) with the rare codon expressing plasmid pRARE2 (Novagen).Expression was induced with IPTG in TB (terrific broth) medium. Theoverexpressed protein was purified initially as described by Ye and byTarus for the clarification and ion-exchange steps, but in a final stepthe fusion protein was purified by affinity on Heparin Sepharose, and bygel filtration (Hi Prep 26/60 Sephacryl S-300) as described in thepatent application PCT/EP2013/076289 filed on Dec. 11, 2013.

To express the NPm-IMX313T protein, the plasmid expressing NP wasmodified in two steps. First, the monomerizing mutations were introducedas in Example 3, using the oligonucleotide primers IMX1287 (5′ccattctgccgcatttgCagatctaagag 3′—SEQ ID NO: 14) and IMX1288 (5′CAAAAGGGAGATTTGCCTGTACTGAGAAC 3′—SEQ ID NO: 15). In a second step, the6-His-tag was replaced by the IMX313T coding sequence, using the sameoligonucleotide primers as in Example 4: IMX1289 (SEQ ID NO: 10) andIMX051 (5′ GTAGAAACAAGGGTATTTTTCTTtattaggagcgacggcgacgc 3′—SEQ ID NO:16). The PCR product was then inserted in place of the 6-His-tag asdescribed by Geiser.

The NPm-IMX313T fusion protein was expressed in the same manner andstrain as the NP protein, and purified using the same chromatographicsteps.

Example 8—Immunisations

Immunisations of mice were then performed to compare the immunogenicityof NPm-IMX313T, with or without formulation with the AddaVax adjuvant(Invivogen). NP protein, with or without formulation with the AddaVaxadjuvant, was used as a control.

To this end, 4 groups of (five) female BALB/c mice were immunizedsubcutaneously twice, with a 14 day interval, using 20 μg of eachprotein per injection. The induction of antigen-specific T-cellresponses were measured by ELISPOTs, using splenocytes, on day 28.Purified spleen CD4+, CD8+ and Total T cells isolated from the immunizedmice were co-cultured with NP protein or Influenza A NP (366-374)peptide. Pre-immune and day 28 antibody responses were measured byELISAs with NP as antigen.

Example 9—IMX313T is not Degraded by Proteases on Passage ThroughSecretion Pathways

The results obtained by DNA immunizations with plasmids containingIMX313T strongly suggest that the tail of the molecule is not cleaved byproteases as it passes through the secretion pathway, where proteasesare abundant. To examine this question more directly, transfection ofCHO K1 cells was undertaken with the pcDNA3 plasmid used to expressNPm-IMX313T in vivo. The transfection was carried out as describedelsewhere (Krammer).

Eighteen to twenty-four hours later, the supernatants of the transfectedcells were recovered by centrifugation, and filtered before being loadedonto a Heparin Sepharose column, as described in the patent applicationPCT/EP2013/076289 filed on Dec. 11, 2013.

A small “peak C” was seen which proved on SDS-PAGE and Western Blottingto contain the protein NPm-IMX313T.

Example 10—Production of Recombinant NPm-IMX313P Protein

To express the NPm-IMX313P protein, the plasmid expressing NPm-IMX313Twas modified by substituting the IMX313P gene in place of the IMX313Tgene, by exchanging a restriction fragment (Pml I-Hind III) from aplasmid encoding IMX313P in place of the corresponding fragment in theplasmid encoding the NPM-IMX313T protein. Then the fusion protein wasexpressed and purified as in Example 7. FIG. 19 shows the purifiedprotein; the principal band is the monomer, but oligomeric forms arealso visible (on the overloaded gel) above the principal band.

Example 11—Production of Hyperimmune Antisera to IMX313P

A group of five female BALB/C mice were immunized intramuscularly sixtimes, at 14 day intervals, with the IMX313P protein using 50 μg perinjection.

Sera were tested for IgG antibodies by using a modified ELISA method.Protamine sulfate Grade X (Sigma), IMX313 or IMX313P were used to coatthe wells of the microplate to capture antibodies. The detectionantibodies were goat-anti-mouse IgG-HRP (Sigma), which was reacted withhydrogen peroxide to produce the absorbance readings at 405 nm.

All sera of mice immunized with IMX313P exhibited high titers of IgGantibodies to IMX313P, and some antibodies which cross-reacted withIMX313; but none cross-reacted with Protamine (FIG. 20).

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The invention claimed is:
 1. An immunogenic composition comprising: afusion protein comprising at least one variant of an influenzanucleoprotein antigen (NP) and a variant of the carrier protein ofsequence SEQ ID NO: 1 comprising a C-terminal substitution of at leastone positively-charged peptide having the sequence ZXBBBBZ SEQ ID NO: 3wherein (i) Z is any amino acid or is absent, (ii) X is any amino acidand (iii) B is an arginine (R) or a lysine (K), wherein the compositiondoes not have an adjuvant.
 2. The composition according to claim 1wherein the at least one variant of an influenza nucleoprotein antigenis a monomeric antigen.
 3. The composition according to claim 1 whereinthe at least one variant of an influenza nucleoprotein antigen is froman Influenza strain A, B or C.
 4. The composition according to claim 3wherein the at least one variant of an influenza nucleoprotein antigenis encoded by the sequence SEQ ID NO:
 2. 5. The composition according toclaim 1 wherein the variant of the carrier protein is the sequence SEQID NO: 4 or SEQ ID NO:
 5. 6. The composition according to claim 2wherein the at least one variant of an influenza nucleoprotein antigenis from an Influenza strain A, B or C.
 7. The composition according toclaim 3 wherein the variant of the carrier protein is the sequence SEQID NO: 4 or SEQ ID NO:
 5. 8. The composition according to claim 1,wherein the at least one variant of an influenza NP antigen comprises asignal peptide.
 9. A method for inducing an immune response againstinfluenza disease in a human or animal in need thereof, comprisingadministering in said human or animal bodies an immunogenic compositioncomprising: a fusion protein comprising at least one variant of aninfluenza nucleoprotein antigen (NP) and a variant of the carrierprotein of sequence SEQ ID NO: 1 comprising a C-terminal substitution ofat least one positively-charged peptide having the sequence ZXBBBBZ SEQID NO: 3 wherein (i) Z is any amino acid or is absent, (ii) X is anyamino acid and (iii) B is an arginine (R) or a lysine (K), wherein thecomposition does not have an adjuvant.
 10. The method according to claim9, wherein the at least one variant of an influenza nucleoproteinantigen (NP) is a monomeric antigen.
 11. The method according to claim9, wherein the administration is intramuscular, subcutaneous, intranasalor intradermal.
 12. A method for increasing T-cell immunogenicity of anucleoprotein antigen (NP) in a human or animal in need thereof,comprising administering in said human or animal bodies an immunogeniccomposition comprising: a fusion protein comprising at least one variantof an influenza nucleoprotein antigen (NP) and a variant of the carrierprotein of sequence SEQ ID NO: 1 comprising a C-terminal substitution ofat least one positively-charged peptide having the sequence ZXBBBBZ SEQID NO: 3 wherein (i) Z is any amino acid or is absent, (ii) X is anyamino acid and (iii) B is an arginine (R) or a lysine (K), wherein thecomposition does not have an adjuvant.
 13. The method according to claim12, wherein the at least one variant of an influenza nucleoproteinantigen (NP) is a monomeric antigen.
 14. The method according to claim12, wherein the administration is intramuscular, subcutaneous,intranasal or intradermal.